Novel therapeutic uses of glucan

ABSTRACT

A process for the production of β-(1,3)(1,6) glucan from a glucan containing cellular source is described, together with compositions and uses/methods of treatment involving glucan. The process of the invention comprises the steps of: (a) extracting glucan containing cells with alkali and heat, in order to remove alkali soluble components; (b) acid extracting the cells of step (a) with an acid and heat to form a suspension; (c) extracting the suspension obtained of step (b) or recovered hydrolyzed cells with an organic solvent which is non-miscible with water and which has a density greater than that of water separating the resultant aqueous phase, solvent containing phase and interface so that substantially only the aqueous phase comprising β-(1,3)(1,6) glucan particulate material remains; wherein the extraction with said organic solvent provides separation of glucan subgroups comprising branched β-(1,3)(1,6)-glucan, and essentially unbranched β-(1,3) glucan which is associated with residual non-glucan contaminents; and (d) drying the glucan material from step (c) to give microparticulate glucan.

FIELD OF INVENTION

[0001] The present invention relates to a process for the extraction ofa naturally occurring carbohydrate (glucan) from microorganisms as wellas the glucan produced by this process. The invention also relates tonovel therapeutic uses of glucan.

BACKGROUND TO THE INVENTION

[0002] Glucan is a generic term referring to an oligo- or polysaccharidecomposed predominantly or wholly of the monosaccharide D-glucose.Glucans are widely distributed in nature with many thousands of formspossible as a result of the highly variable manner in which theindividual glucose units can be joined (glucosidic linkages) as well asthe overall steric shape of the parent molecule.

[0003] The glucan referred to in this invention typically is a linearchain of multiple glucopyranose units with a variable number ofside-branches of relatively short length. The glucosidic linkages arepredominantly (not less than 90%) β-1,3 type with a lower number (notgreater than 10%) of β-1,6 type linkages; the β-1,3 linkages form thebulk of the backbone of the molecule, while the β-1,6 linkages occurpredominantly in the side-branches. The chemical name of this form ofglucan is poly-(1,3)-β-D-glucopyranosyl-(1,6)-β-D-glucopyranose. Glucanis a well described molecule.

[0004] This form of glucan is found principally in the cell wall of mostfungi (including yeasts and moulds) and in some bacteria. Glucan, incombination with other polysaccharides such as mannan and chitin, isresponsible for the shape and mechanical strength of the cell wall. Theglucan typically accounts for approximately 40% to 50% of the weight ofthe cell wall in these cells.

[0005] The chemical structure of fungal cell wall glucan has beenstudied in detail, with the following sentinel articles beingincorporated herein by reference—Bacon et al (1969); Manners et al(1973).

[0006] Fungal cell wall glucans have long been used in industry,particularly the food industry, usually in a semi-purified form. Theiruses have included use as stabilizers, binders, thickeners and surfaceactive materials.

[0007] It also has been known for some forty years that fungal cell wallglucans are biologically active, exerting a number of effects on thereticuloendothelial and immune systems of animals. The outstandingbiological effect in this regard is their ability to stimulate nonspecifically the activity of the body's primary defence cells—themacrophage and the neutrophil. This is thought to be due to receptors toβ-1,3 glucan displayed on the surface of these cells (Czop and Austen,1985). The interaction between glucan and its receptor producing suchstimulatory effects as enhanced phagocytosis (Riggi and Di Luzio, 1961),increased cell size (Patchen and Lotzova, 1980), enhanced cellproliferation (Deimann and Fahimi, 1979). enhanced adherence andchemotactic activity (Niskanen et al, 1978), and production of a widerange of cytokines and leukotrienes (Sherwood et al. 1986, 1987).

[0008] The aforementioned biological responses to fungal cell wallglucan have been reported to result in a number of clinical effectsincluding: enhanced resistance to infections with fungi (Williams et al,1978), bacteria (Williams et al, 1983), viruses (Williams and Di Luzio,1985). protozoa (Cook et al, 1979) following systemic application:enhanced antitumour activity following systemic application(Williams etal, 1985) or intralesional application (Mansell et al, 1975); andenhanced immune responsiveness following systemic application (Maeda andChihara, 1973). It will be readily seen that these clinical effects arehighly beneficial and important and represent an opportunity to developnovel pharmaceutics based on fungal cell wall glucans, suchpharmaceutics having potentially wide application in both veterinary andhuman medicine.

[0009] Of the various fungal cell wall glucans tested, that from theveast Saccharomyces cerevisiae has proven to be acceptable in terms ofefficacy and safety as an immune stimulant in animals and humans.Hereinafter this will be referred to as Saccharomyces cerevisiae(“Sc”)-glucan. Predominantly or wholly β-1,3 glucans from other fungi,bacteria or plants from the Graminaceae family have been shown to beimmunostimulatory in animals but compared to Sc-glucan either are not aspotent or if they do have comparable or greater potency then that isusually associated with a higher level of undesirable side-effects.

[0010] Sc-glucan has been shown to be biologically active as an immunestimulant in animals in various forms. These include (a) a largemolecular weight (typically greater than 3×10⁶ d), water-insoluble,microparticulate form, or (b) smaller molecular weight (typically lessthan 500,000 d) forms which are dispersible or soluble in water.Water-solubility is described as being achieved either through cleavageof the large microparticulate glucan form to smaller molecules usingprocesses such as enzymatic digestion or vigorous pH adjustments, or bycomplexing to salts such as amines, sulphates and phosphates. Theprincipal advantage of the smaller, water-soluble form vs the largermicroparticulate form is that it is safer when given by parenteralroutes of administration such as intravenously. Also, it is likely thatthe smaller sized molecules are more bio-available on a molar basis.

[0011] To date it has neither been technically possible nor economicallyfeasible to synthesise glucan on a commercial basis. Thus preparation ofcommercial quantities of β-1,3 glucan for therapeutic uses requires thatit be extracted from fungi, bacteria, algae or cereal grains.

DESCRIPTION OF THE PRIOR ART

[0012] A number of different processes are described for the preparationof Sc-glucan for pharmaceutical use. A common feature of these differentprocesses is the extraction of microparticulate glucan as the primarystep; the glucan is either then used in the final therapeuticformulation in that microparticulate form or is further processed to asmaller molecular weight material (“soluble glucan”) by modification ofits chemical and/or spatial structures.

[0013] (i) Microparticulate Glucan

[0014] The extraction of Sc-glucan from whole yeast cells depends on thefact that the bulk of the cell wall glucan is insoluble in water, strongalkali, acid and organic solvents whereas all other cell wall componentsare soluble in one or more of these solutions.

[0015] The essential principles of extraction of Sc-glucan are (i) lysisof the yeast cell to allow the intact cell walls to be separated fromthe less dense cytoplasmic contents, and (ii) subsequent or concomitantdissolution of unwanted wall components such as other carbohydrates(glycogen, mannan, glucosamine), lipids and proteins using variouscombinations of water, alkali, acid and organic solvents. It ispreferred in such processes that the three-dimensional matrix structureof the cell wall remains unaltered and intact as a cell wall skeleton(also known as a “cell sac”), comprised predominantly ofβ-(1,3)(1,6)-glucan. The cell wall skeletons characteristically arespherical, hollow structures of approximately 4 to 20 u diameter andwith a molecular weight of between approximately 1,000,000 to 3,000,000daltons and they are insoluble in water. This end-product is termedmicroparticulate Sc-glucan.

[0016] A number of methods of extraction of microparticulate Sc-glucanare known, although all are essentially variations of a common method.The described methods entail the following steps.

[0017] 1. Contact of whole yeast cells with strong alkali solution (pH12 to 14). This effects lysis of the cells and dissolution of most ofthe non-glucan components except lipids. This step is uniformly rigorousin all described processes. The contact usually is repeated two to threetimes using fresh batches of alkali and heat also usually is applied tospeed the reaction time.

[0018] 2. The cells then are exposed to acid (pH 1 to 5) with heat toeffect dissolution of certain residual non-glucan components and toeffect some hydrolysis of the glycosidic linkages, principally the β-1,6linkages in the side brances and to a minor extent β-1,3 linkages in theglucan backbone side-branches. The rigour of this step variesconsiderably between the known processes of relatively mild acidtreatment where the conformational changes are minimal and many of theside-branches are retained, through to extensive acid treatment wherelittle or no side-branches remain and which permits hydration of thehelical glucan coils during subsequent steps to convert to awater-soluble form.

[0019] 3. Contact of the cell residue with alcohol and heat with orwithout additional subsequent exposure to solvents, particularly etheror petroleum ether to effect removal of lipids.

[0020] See, for example, Hassid et al (1941), Manners (1973) et al, DiLuzio (1979), and U.S. Pat. Nos. 4,810,694 and 4,992,540.

[0021] Prior art methods for the production of microparticulate glucanmay be regarded as disadvantageous in one or more respects. Theseinclude poor yield (such as less than about 5% w/w), low purity (such asless than about 90% purity), extended processing time, significant wasteproduction, and high cost.

[0022] (ii) Soluble Glucan

[0023] Microparticulate Sc-glucan is water insoluble due to the tightlybound triple helical carbohydrate coils which resist hydration.

[0024] There are two principal purposes to seek to solubilize Sc-glucan.The first reason is the risk of microembolization associated with theinjection of microparticulate glucan by intravenous or other parenteralroutes. The second reason is that a reduction in molecular weight of theSc-glucan might reasonably be expected to be associated with increasedbiological efficacy due to greater bioavailability of the glucanmolecules.

[0025] Solubilization of microparticulate glucan can be achieved invarious ways.

[0026] One way is to expose the glucan to a specific enzyme.β-1,3-glucosidase which cuts the long linear chain into shorter lengths.The disadvantage of this method is that the enzymic digestion process isdifficult to control and can result in excessive hydrolysis of theglucan molecule to monosaccharides or oligosaccharides which lackimmunostimulatory activity.

[0027] Another way is to attach charged groups such as phosphate (U.S.Pat. Nos. 4,739,046; 4,761,402), sulphate (Williams et al, 1991) andamine (U.S. Pat. No. 4,707,471) which permit hydration of the molecule.Both phosphorylated (U.S. Pat. No. 4,761,042) and sulphated (Williams etal, 1991) Sc-glucans retain their immunostimulatory activity and arehighly water soluble. A disadvantage of these methods is that of anadditional step of complexity in processing operations, which may addconsiderably to overall manufacturing cost.

[0028] A third approach to solubilization is by sequentialalkali/acid/alkali hydrolysis. This was first demonstrated by Bacon etal (1969) who showed that microparticulate Sc-glucan extracted in thetraditional manner by repeated NaOH exposures followed by an acid wash,almost completely dissolved when subsequently exposed to 3% NaOH at 75°C. This phenomenon is described again in PCT/US Application No 90/05041whereby microparticulate Sc-glucan following exposure to acetic acid orformic acid is exposed to IN NaOH for one to two hours at 80° C. to 100°C. The resultant glucan is of widely heterogenous molecular weight witha high polydispersity index associated with the presence of glucanmolecules varying in size from approximately 5,000 d up to approximately800,000 d. That patent application describes further purification bydiafiltration of the hydrolyzed glucan to isolate glucan molecules ofdefined molecular weight from the heterogenous molecular weight speciesproduced, and the use of various resins to remove contaminatingproteinaceous and lipid components.

[0029] The present invention insofar as it is concerned with processesfor the production of glucan, whether in microparticulate ornon-particulate form (“soluble”), seeks to overcome one or more of theproblems/deficiencies of prior art processes for the production ofglucan.

[0030] In addition, as described hereinafter, this invention is alsoconcerned with novel therapeutic uses of glucan, whether produced by themethod herein, or other methods known in the prior art.

SUMMARY OF THE INVENTION

[0031] In accordance with a first aspect of this invention there isprovided a process for production of β-(1,3)(1,6) glucan from a glucancontaining cellular source which comprises the steps of:

[0032] (a) extracting glucan containing cells with alkali and heat inorder to remove alkali soluble components;

[0033] (b) acid extracting the cells obtained from step (a) with an acidand heat to form a suspension;

[0034] (c) extracting the suspension obtained from step (b) or recoveredhydrolyzed cells with an organic solvent which is non-miscible withwater and which has a density greater than that of water and separatingthe resultant aqueous phase solvent containing phase and interface sothat substantially only the aqueous phase comprising glucan particulatematerial suspended in water remains: wherein the extraction with saidorganic solvent provides separation of glucan subgroups comprisingbranched β-(1,3)(1,6)-glucan, and essentially unbranched β-(1,3) glucanwhich is associated with residual non-glucan contaminents: and

[0035] (d) drying the glucan material from step (c) to give particulateglucan.

[0036] In order to produce a soluble glucan, step (d) of the aboveprocess is omitted and the pH of the solvent extracted aqueous phasecomprising glucan particulate material is raised from an acidic pH, to abasic pH so as to effect solubilization of the glucan particles. Thisstep is carried out at a temperature below about 60° C., preferablybetween about 2° C. to about 25° C. more preferably between about 2° C.to about 8° C., for a time sufficient to achieve solubilization of theglucan particles. Alternatively, soluble glucan may be prepared bysuspending the particulate glucan of step (d) in an aqueous alkalisolution so as to effect solubilization of the glucan particles.Temperate conditions are set out above.

[0037] The pH of the solubilized glucan may then be adjusted as requiredto give a pharmaceutical product.

[0038] In another aspect this invention is directed to the use of glucanfor the manufacture of a medicament for the treatment of skin ulcerationor bone fracture or the enhancement of fixation of implanted orthopaedicdevices, or the prevention/treatment of ultraviolet light induced skindamage.

[0039] In a further aspect this invention is concerned with a method forthe treatment of skin ulceration or bone fracture or the enhancement offixation of implanted orthopaedic devices, or the prevention/treatmentof ultraviolet light induced skin damage, which comprises administeringto a subject glucan in association with one or more pharmaceutically orveterinarily acceptable carriers or excipients.

[0040] In another aspect this invention is concerned with an agent forthe treatment of skin ulceration or bone fracture or the enhancement offixation of implanted orthopaedic devices, or for theprevention/treatment of ultraviolet light induced skin damage whichcomprises glucan optionally in associate with one or morepharmaceutically acceptable carriers or excipients.

DETAILED DESCRIPTION OF THE INVENTION

[0041] The process described in detail hereafter sets out the productionof β-(1,3)(1,6) glucan from a cellular glucan source, which is suitablefor a variety of pharmaceutical purposes.

[0042] In a first aspect the invention is concerned with a process forthe production of glucan from a glucan containing cellular source. Thisprocess comprises the steps of:

[0043] (a) extracting glucan containing cells with alkali and heat, inorder to remove alkali soluble components;

[0044] (b) acid extracting the cells of step (a) with an acid and heatto form a suspension;

[0045] (c) extracting the suspension obtained of step (b) or recoveredhydrolyzed cells with an organic solvent which is non-miscible withwater and which has a density greater than that of water and separatingthe resultant aqueous phase, solvent containing phase and interface sothat substantially only the aqueous phase comprising glucan particulatematerial remains; wherein the extraction with said organic solventprovides separation of glucan subgroups comprising branchedβ-(1,3)(1,6)-glucan, and essentially unbranched β-(1,3) glucan which isassociated with residual non-glucan contaminants: and

[0046] (d) drying the glucan material from step (c) to give particulateglucan.

[0047] While yeast cells generally and the yeast strain Saccharomycescerevisiae in particular are the preferred source of the glucanaccording to this invention, any other cells such as fungi or bacteriacontaining glucan with the properties described herein may be used. Awide range of other yeast and fungal strains can be used in the presentprocess and the following types are included by way of example:Sclerotium spp, Shizophyllum spp, Pichia spp, Hansenula spp, Candidaspp, Saccharorryces spp, Torulopsis spp.

[0048] In the case of Saccharomyces cerevisiae the yeast may be grownspecifically for the purpose of extraction of Sc-glucan or may be from acommercial source such as yeast manufactured for the baking industry orspent yeast from the brewing industry.

[0049] The first step according to the process of the present inventioninvolves treatment of the yeast cells with alkali and heat to effectcytolysis and hydrolysis of the cytoplasmic components and predominantcell wall components including mannan, chitin (glucosamine), proteinsand glycogen. This treatment (which may also be referred to asextraction or hydrolysis) releases non-glucan components into theaqueous phase so that they might readily be separated by a process suchas centrifugation from the intact cell walls comprising largely glucan.The extent of non-glucan component removal can be readily assessed bystandard analytical techniques, such as those described in U.S. Pat. No.4,992,540.

[0050] The alkali extraction step may be carried out in aqueoushydroxide of from about 2% to about 6% concentration (w/v), such asbetween 3% and 4% (w/v). Sodium hydroxide or potassium hydroxide findparticular application because of their availability and relatively lowcost. However, any other strong alkali solution which has suitablesolubility characteristics, for example, calcium hydroxide or lithiumhydroxide, can be used. The yeast is left in contact with the alkali fora time sufficient to remove alkali soluble non-glucan components.Non-glucan components are removed more rapidly at higher temperatures.The digestion may be carried out at temperatures of from about 50° C. toabout 120° C., requiring exposure times to the alkali of between fifteenminutes and sixteen hours. During alkali exposure, the process ofcytolysis and dissolution of non-glucan components may be facilitated byvigorous mixing of the yeast suspension using appropriate methods suchas by example a stirring apparatus or an emulsifying pump.

[0051] Repeat exposure of the yeast cells to fresh batches of alkalisolution assists in removing non-glucan material, particularly protein,from the disrupted yeast cells. The number of alkali treatments is notlimiting on the invention. However, the process should be repeated untilit is apparent that the cells have been lysed and the majority, ofnon-glucan alkali soluble components extracted. This can be confirmed byvisual or chemical analysis (such as by gas chromatography/massspectrometry). Treatments using low strengths of hydroxide solution andlow temperatures of alkali exposure generally may require increasednumbers of separate alkali exposures. By way of example, alkalitreatment may be repeated from one to six times.

[0052] In one embodiment of the present invention in relation to thealkali digestion phase, dried commercial Saccharomyces cerevisiae issuspended to 10% w/v in sodium hydroxide at a strength of between 3% and4% and at temperatures of between 80° C. and 100° C. It has been foundthat three alkali treatments are typically required for a high purityproduct. Following each separate alkali exposure, the disrupted yeastcells and the supernatant solution are separated by any method which isknown to this art including, for example, filtration, centrifugation orchromatography. These separation techniques are referred to by way ofexample only and are not limiting to the process of the presentinvention.

[0053] The next step in the process involves the exposure of thealkali-insoluble cell wall sacs to acid, generally at a pH from about2.0 to 6, preferably between 3.5 to 4.5. This procedure dissolves someresidual contaminants such as mannan and chitin. However, the principalreason for this step is to induce conformational alterations to theglucan molecule. The principal alteration is a reduction in the numberof β-1,6 side-branches (Table 1). In native cell wall Sc-glucan, theproportions of glycosidic linkages is approximately 90% β-1,3 and 10%β-1,6. Acid hydrolysis removes the β-1,6 side-branches with the degreeof hydrolysis being related directly to the vigour of the acidtreatment; strong acid treatment (low pH and high temperature, such aspH less than 2 and temperatures above about 100° C.) can effectivelyremove all side-branches whereas less vigorous treatment will leaveβ-1,6 linkages in the proportions of between approximately 1% and 8%.TABLE 1 Effect of acid exposure (phosphoric acid, ph 4.5, 100° C. 30minutes) on the chemical composition of alkali insoluble Sc-glucan asmeasured by gas chromatography-mass spectroscopy. Pre-acid Post-acidMannan (% w/w monosaccharides) 0.5 0 β-glycosidic linkages (mol %): 1,354.2 94.4 1,4 7.1 0 1,3,4 0.7 0.2 1,2,3 2.2 0.5 1,3,6 5.6 2.2 1,6 9.7 01,4,6 0.8 0 1,2,3,4 1.5 0 1,3,4,6 1.9 0 1,2,3,6 0.4 0 Terminal-glc 6.42.9 glucitol hexaacetate 10.8 0

[0054] It is known in the art that the degree of branching ofβ-1,3-glucan molecules has an important influence on biologicalfunction. For example, it is known that highly branched glucans such aslentinan induce pro-inflammatory effects in addition toimmunostimulatory effects and that the pro-inflammatory effects may beassociated with adverse clinical side-effect; unbranched Sc-glucans suchas those described in U.S. Pat. Nos. 4,739,046. 4,761,402 and 4.7707,471or Sc-glucan with reduced branching such as that detailed in PCT/USPatent No. 90/05041 are known to avoid or to greatly diminishpro-inflammatory effects and therefore be more desirable therapeuticagents clinically, Hitherto, however, the structure/functionrelationship in terms of immunostimulatory capacity and promotion oftissue repair in particular has not been defined. The inventors havedefined the optimal degree of branching by comparing the efficacy ofdifferently branched glucan preparations in an animal wound healingmodel. For example, a full-thickness surgical skin incision may be madein experimental animals such as laboratory rats. Glucan is applied tothe wound immediately following wounding and the wound then allowed toheal. Seven days later the degree of healing is tested by determiningthe amount of force required to separate the apposing wound edges(referred to as ‘wound breaking strength’). The results of thisexperiment are summarised in Table 2. It can be seen that where thedegree of branching is measured in terms of the proportion ofβ-1,3:β-1,6 linkages, both a low proportion (90%:10%) as for nativeglucan and a high proportion (100%:0%) are less effective In thepromotion of dermal wound repair than moderately-branched (98%:2% or96%:4%) glucan. TABLE 2 Tensile strength of rat skin wounds (day + 7)following application of micro-particulate Sc-glucans with differentratios of β-1,3 to β-1,6 glycosidic linkages. Wound tensile strength (g)Treatment n β-1,3:β-1,6 linkages mean (SD) No glucan 16 — 202 (37)Glucan 8 90%:10% 252 (45) Glucan 12 96%:4%  358 (49) Glucan 9 98%:2% 339 (38) Glucan 10 100%:0%  285 (52)

[0055] The nature of the acid used in the acid exposure step isgenerally unimportant. Preferably, the acid is employed to provide a pHof the resultant yeast suspension from about pH 2.0 to about 6.0. morepreferably from about pH 3.5 to about 4.5. Suitable acids includehydrochloric, acetic, formic and phosphoric acids.

[0056] The process of acid hydrolysis is aided by heating.

[0057] The extent of acid treatment, namely pH, temperature and timedepends on the degree of β-1,6 content sought in the glucan product. Inorder to produce a glucan product generally containing from 2% to 4%β-1,6 linkages, the pH of the solution is selected to be in the range ofabout 2 to about 6, temperature is generally between about 50° C. andabout 100° C., and the time of reaction from about fifteen minutes toabout sixteen hours. The extent of β-1,6 linkages in the hydrolyzedglucan can be readily determined by standard analytical techniques suchas nuclear magnetic resonance (NMR) analysis.

[0058] Following the acid exposure stage, the yeast cells predominantlyare in the form of isolated cell wall sacs.

[0059] In prior art methods of Sc-glucan preparation it has beenproposed to expose acid extracted glucan containing cells (cell sacs)with alcohol, petroleum ether or diethyl ether, to selectively dissolveremaining non-glucan components. In contrast, it has been found by theinventors that extracting the acidified glucan containing cells with anorganic solvent which is non-miscible with water, that is, has a densitygreater than 1 g/cm³, is particularly and unexpectedly advantageous.Specifically, a single extraction step with such a solvent provides afine discrimination between glucan and non-glucan components, and allowsready separation of glucan subgroups comprising branched glucancontaining both β-1,3 and β-1,6 linkages (which partitions into theaqueous phase) and which is essentially free of non-glucan components(Table 3), and glucan comprising essentially unbranced β-1,3 linkagesonly and which is associated with residual non-glucan membranecomponents such as chitin and protein (which partitions at the interfacebetween the aqueous and organic phase). TABLE 3 Effect of chloroformextraction on the chemical composition of alkali/acid treated Sc-glucan. Chemical composition (% w/v) Glyco- Glucan Mannan Protein Chitingen Lipids Prechloroform 85.5 0.5 1.4 2.1 4.3 5.6 treatmentPostchloroform 98.5 <0.1 0.3 0.2 0.4 — treatment

[0060] The branched β-(1,3)(1,6) glucan subgroup which partitions intothe aqueous phase may contain minor or trace amounts of unbranched β-1,3glucan (less than about 5%, generally less than about 2%, morespecifically less than about 0.5% (w/w)) and trace amounts of non-glucancontaminents. It may thus be regarded as essentially branchedβ-(1,3)(1,6) glucan which is free of other glucan and non-glucancomponents. The unbranched β-(1,3) glucan subgroup which is associatedwith non-glucan contaminants and which partitions into the interfacebetween the aqueous phase and organic phase can be readily removed. Itmay contain very minor or trace amounts of branched β-(1,3)(1,6) glucan(generally less than about 1.3% (w/w)) and hence is considered to beessentially unbranched.

[0061] Unbranched β-(1,3) glucan may comprise up to 20% of total glucancontent (w/w) following alkali/acid/solvent treatment, the remaindercomprising branched β-(1,3)(1,6) glucan.

[0062] Branched β-(1,3)(1,6) glucan is the most potent biologicallyactive form of glucan in terms of wound healing as shown in Table 4.TABLE 4 Tensile strength of rat skin wounds (day + 7) followingapplication of Sc-glucans recovered from either the aqueous or interfacephase following chloroform extraction. Wound tensile strength (g)Treatment n Post-chloroform phase mean (SD) No glucan 12 — 185 (21)Glucan 14 Aqueous 345 (57) Glucan 8 Interface 267 (59)

[0063] Thus it can be readily appreciated, particularly in terms ofefficacy of promotion of dermal wound healing and the production of pureglucan molecules, that there is much potential therapeutic benefit inseparating the two glucan sub-groups by chloroform extraction(representative of solvents having a density greater than 1).

[0064] Solvents which may be used include chloroform (δ=1.48 g/cm³).methylchloroform (δ=1.33), tetrachloroethane (δ=1.5953 g/cm³).dichloromethane (δ=1.325), and carbon tetrachloride (δ=595 g/cm³).Preferably the solvent is volatile to allow ease of removal of anyresidual. Chloroform is particularly preferred.

[0065] For convenience of description the description hereafter willrefer to the use of the preferred solvent chloroform. The invention isnot so limited, and any solvent having the requisite density may be usedin the invention.

[0066] The chloroform extraction may be performed in the followingmanner. The acidified aqueous suspension containing microparticulateglucan may be reacted directly with chloroform in the approximate ratioof chloroform:aqueous cell suspension of between 1:10 and 5:1,preferably 1:4. The yeast cells may comprise (by volume) between about1% and about 90% of the aqueous suspension, such as between about 30%and 50%. It has been found that the process of extraction withchloroform is not facilitated by heat and preferably is carried out atroom temperature. The chloroform and aqueous phases are mixed vigorouslyusing standard methods including, for example, stirring apparatuses oran emulsifying pump so as to effect good contact between the chloroformmicelles and the yeast cells. The duration of mixing is a function ofthe volume of the suspension and the stirring or mixing capacity of thestirring or mixing apparatus. An example by way of illustration is thatan emulsifying pump with a pumping capacity of 100 L per minute would berequired to mix a suspension volume of 500 L for about ten minutes.

[0067] A notable feature of the chloroform extraction step is that theyeast material changes nature both in colour (converting from alight-gray colour to a white colour) and in form (converting from amaterial with typical cellular characteristics (cell sacs) in suspensionto a flocculent particulate material). The bleaching and flocculatingeffects observed as a result of contact with chloroform (and othersolvents having the requisite density referred to above), have not beenobserved with other organic solvents which have a density less than 1g/cm³. Solvents which have been tested in this regard include acetone,diethyl ether, petroleum ether, methylene dichloride, ethyl acetate,ethanol, methanol and butanol.

[0068] Following chloroform exposure and mixing such as between aboutfive and ten minutes, the suspension is allowed to settle and quicklyseparates into three distinct phases—a lower organic phase, an upperaqueous phase, and an interface between those two phases which iscoloured gray. The three phases are well differentiated and readilyseparated. The organic phase is slightly opaque and contains lipids butno glucan. The aqueous phase contains glucan particles suspended inwater. The interface contains a mixture of glucan, protein, and chitinand lipids. When analyzed by NMR, the glucan in the aqueous phasecontains a mixture of β-1,3 and β-1,6 glycosidic linkages in theapproximate ratio of 95% to 98%:2% to 5% respectively. The glucan in theinterface phase contains predominantly unbranched β-1,3 glycosidiclinkages (generally 98 to 100% β-1,3:0% to 2% β-1,6. Effectiveseparation of branched β-1,3 glucan unbranched glucan and non-glucancontaminants is achieved.

[0069] This separation of glucan particles based on their level ofnon-glucan contaminants has been found only with solvents having thedensity mentioned above, and not with other commonly available organicsolvents having a density less than 1 g/cm³. Without being bound by anyparticular theory the fine discrimination in separating glucan speciesas exemplified by chloroform, may be due to the combination oflipophilic nature of the solvents and their specific density. This mayallow differential separation by weight of cell wall glucan moleculeswhich are associated with other carbohydrates and non-carbohydrates. Theglucan and non-glucan molecules in this interface phase can be separatedsubsequently by evaporation of the chloroform followed by contact of theresidue with ether and ethanol to effect dissolution of the non-glucancomponent, leaving essentially unbranched β-1,3 glucan.

[0070] The aqueous glucan suspension collected following the specificsolvent exposure step may be boiled briefly to effect complete removalof any residual solvent and the glucan particles then dried by standardmethods including for example, freeze-drying, heating, air-drying orspray-drying. The final product is a slightly off-white, flocculentpowder comprising particles of Sc-glucan with a diameter typically ofbetween about 1 u up to 10 u with a median diameter of about 3 u (suchparticles may be referred to as microparticulate glucan). The powder maybe milled using standard procedures (hammer milling or ball milling) togive particles of desired size.

[0071] The separation of predominanly branched and uncontaminatedglucan, from relatively unbranched glucan associated with non glucancomponents, is not achieved where glucan particles are reacted withalcohol prior to reaction with a solvent have density greater than 1,such as chloroform. This is an unexpected finding.

[0072] Prior art description of the use of organic solvents to removelipids from particulate glucan preparations failed to appreciate thediscriminating effects of solvents having a density greater than 1 inseparating predominantly branched, uncontaminated glucan frompredominantly unbranched contaminated glucan. This invention may thus beregarded as a selection which confers substantial advantage as discussedabove.

[0073] The microparticulate Sc-glucan produced by this process can beused as a therapeutic in this form. Some examples of use are applicationfor repair of tissues such as skin and bone and bowel where themicroparticulate Sc-glucan is applied in formulations such as a powderor cream or lotion or can be used in wound dressings such as bandages orhydrocolloid dressings. Conventional topical formulations may beutilized as are well known in the art and described hereafter.

[0074] The process of the invention described above gives rise to a highpurity product, having a highly potent bioactivity (as it may compriseglucan having only β-1,3 and β-1,6 linkages) which is achieved withshort processing time, and high yield. Table 5 demonstrates this bycomparing glucan produced according to this invention with glucanprepared according to the procedures of Hassid et al (1941), Di Luzio etal (1979), Manners et al (1973), and Jarnas (U.S. Pat. No. 4,992,540).TABLE 5 Comparison of four standard methods of extraction ofmicroparticulate Sc-glucan. Pro- Glu- cessing can Component levels (%w/w) Time Yield Glu- Glyco- Method (days) % can Mannan gen ProteinChitin Hassid 8 7.8 91.7 0.4 4.5 2.9 0.4 et al Di Luzio 12 2.0 98.1 0.30.5 0.7 0.2 et al Manners 18 12.1 73.8 2.0 9.8 8.6 5.8 et al Jamas 2 7.494.6 0.3 3.1 0.8 1.1 et al The 2 7.7 98.5 <0.1 0.4 0.3 0.2 presentinvention

[0075] The process of this invention also provides for the conversion ofparticulate glucan to glucan molecules of smaller molecular weight inthe form of a solution, dispersion or colloid, or gel which would besuitable for pharmaceutical, such as parenteral use. Such material mayshow enhanced bioactivity through the greater availability of glucanligands for cytophilic glucan receptors. These glucan preparations maybe regarded as providing glucan in a soluble form, where glucanparticles dissolve in the aqueous phase to give a visually clearsolution, or are otherwise hydrated to the extent that they form adispersion or colloid, or are in the form of a gel. For convenience,these forms may be referred to as soluble glucan.

[0076] In the prior art it has been proposed to convert particulateglucan to soluble glucan using rigorous heat treatement (generally at75° C. or greater) in the presence of alkali (Bacon et al 1969). Inanother proposal, the particulate glucan was treated with strong acid(90% formic acid) prior to exposure to alkali and heat. These approachessuffer from a number of disadvantages which include the production ofheterogenous glucan products of wide polydispersity which are unsuitablefor pharmaceutical use without size fractionation, relativeinconvenience, high cost, and production of waste materials.

[0077] It has been found by the inventors that the glucan purified asdescribed above is readily solubilised in alkali at low temperatures(particularly between about 2° C. and about 8° C.). In the presentinvention, solvent extraction of acid treated cell wall sacs with asolvent which has a density greater than 1, where glucan partitioningtakes place with subsequent separation and isolation of branchedglucans, enables solubilisation in alkali at low temperatures. It isotherwise not possible to produce soluble glucan having the propertiesdescribed hereafter.

[0078] In order to produce soluble glucan, step (d) of the processdescribed above may be omitted and the pH of the solvent extractedaqueous phase comprising glucan particulate material may be raised froman acidic pH to a basic pH so as to effect solubilization of the glucanparticles. This step is carried out at a temperature below 60° C.,preferably from about 2° C. to about 25° C., more preferably from about2° C. to about 8° C. for a time sufficient to achieve solubilization ofthe glucan particles. Alternatively, soluble glucan may be prepared fromglucan of step (d) of the above process by reacting the particlateglucan with an aqueous alkali solution so as to effect solubilization ofthe glucan, particles. Temperature conditions are again below 60° C., asspecified above.

[0079] An unexpected consequence of the present invention is that afteralkali solubilisation a glucan material having a small polydispersityindex (generally less than about 5, more particularly less than about 3)results. This is highly desirable for pharmaceutical agents.Furthermore, no additional size fractionation steps are required. Thisis contrary to prior art teachings as set out above.

[0080] In one embodiment, microparticulate glucan isolated as describedabove may be suspended in NaOH solution at a strength of between about2% and 10% (pH between pH 10 and pH 14.5) but preferably 5%; thesuspension contains between about 0.1 and about 30% (w/w) glucan, suchas 5%. A particular feature of this reaction step as discussed above, isthat contrary to the known art it does not require prior exposure tostrong acid or applied heat or vigorous agitation; the reaction is foundto occur most advantageously at low temperatures (preferably between 2°C. to 8° C.) and with little or no mixing; the reaction time isgenerally between about one and twenty four hours, such as two hours.Between about 90% to 99% of the glucan particles are converted (throughalkaline hydrolysis) to suspended small molecular weight molecules overthe reaction time. At the conclusion of the reaction the undissolvedparticles are removed by standard methods such as, for example,centrifugation or filtration and the pH of the suspension adjusted theaddition of Hcl (say from pH 8 to pH 10). This soluble glucan may beused as a pharmaceutical product. The glucan solution may then beadjusted to isotonicity by standard methods such as dialysis orultrafiltration.

[0081] The glucan material produced by this method has a molecularweight range between approximately 60,000 to 250,000 with a mean ofabout 140,000 daltons, with a mean polydispersity index of about 2.4.Between approximately 70% and 85% of the glucan molecules are within 15%of the mean molecular weight and it is found that this result is highlyreproducible with different batches. This low polydispersity indexindicates relatively high homogeneity. It is thus entirely suitable foruse as a pharmaceutical. It is found that this material has highbiological potency, as measured, for example, in the promotion of tissuerepair. In a rat dermal wound repair model, this material isapproximately five times as efficacious as microparticulate Sc-glucanwhen compared on an equivalent molar basis (Table 6). TABLE 6 Tensilestrength of rat skin wounds (day + 7) following application of a singletopical dose of 1 mg micro-particulate vs soluble Sc-glucan with 96%(β-1,3) and 4% (β-1,6) linkages. Wound tensile strength (g) Treatment nmean (SD) No glucan 12 196 (23) Micro-particulate glucan 14 356 (47)Soluble glucan 8 432 (69)

[0082] In that experiment the glucans were administered in a lipophiliccream base, but it would be anticipated that this material could be usedas a topical therapeutic in a variety of formulations or could beinjected as a parenteral therapeutic.

[0083] In a strongly alkaline solution, the soluble glucan moleculesoccur principally as triple helices but with little or no polymerisationof independent helical structures. The effect of lowering the pH of theglucan solution is to predispose the glucan molecules to polymerisationleading to gel formation. At a pH below approximately 9.0 there isprogressive polvmerisation of adjacent helical structures. It isobserved that the degree of polymerisation of the glucan molecules isrelated directly to the concentration of the glucan solution. Where theglucan solution is to be diluted and dispersed in a carrier vehicle andit is desirable to rinirnise the degree of polymerisation, theconcentration of the glucan solution is generally less than 10 mg/mL,and preferably no greater than 5 mg/mL prior to adjustement of the pHfrom a strongly alkaline state (around pH 13). In other instances it maybe desirable to have the final glucan solution as a gel and this isachieved if the concentration of the glucan solution prior to pHadjustment is greater than 10 mg/mL (10% w/w) and preferably greaterthan 15 mg/mL (15% w/w). for example up to about 30% w/w. It is foundthat this gel state is a convenient form for topical application,requiring little or no additional formulation.

[0084] It can be seen that the present manufacturing process representsa significant advance over the current state of the art in this field.Compared to other known manufacturing processes, the present processyields an end-product which has greater purity, is manufactured in ashorter time, has greater efficiency of vield, produces a glucanmolecule of distinctive chemical structure, and produces a product ofdesired homogeneity without the necessity of elaborate and expensiveseparation techniques.

[0085] It readily would be appreciated that these advantages lead toconsiderable cost savings, with the availabilitv of a less expensivematerial thus allowing wider application of Sc-glucan as a therapeuticin both veterinary and human medicine than is currently available.

[0086] The applications for which the microparticulate Sc-glucanproduced by the process of the present invention are suitable includethose applications in particular where the risk of direct entry of thematerial to the bloodstream is minimal and these include by way ofexample oral application, topical application, intradernal injection,intramuscular injection, subcutaneous injection, intraperitonealinjection, intrathecal injection, intralesional injection, intratendoninjection, intraligament injection, intraarticular injection, andapplication to fracture sites of bones and cartilage. The therapeuticpurposes include by way of example (a) enhancement of wound repairprocesses in the aforementioned tissues, (b) enhancement of resistanceto infection from bacterial, fungal, viral and protozoal organisms inthe aforementioned tissues, and (c) enhanced local immune responsivenessto carcinogenesis.

[0087] The applications for which the small molecular weight Sc-glucanproduced by the process of the present invention are suitable include byway of example although not being limited to those listed above formicroparticulate Sc-glucan: indeed in these situations the use ofsoluble Sc-glucan may be preferred to that of microparticulate Sc-glucanbecause of various practical considerations such as ease ofadministration or the benefit of administration in a liquid form orbecause of the greater bioavailability of this form. However, smallmolecular weight Sc-glucan has particular indication for thosesituations where penetration of intact tissues (such as trans-epidermalpenetration of intact skin ) is desired or where entry of the materialto the bloodstream may occur inadvertently.

[0088] The Sc-glucans produced by the processes of the present inventioncan be presented in formulations commonly used in the pharmaceutical andcosmetic industries including, for example ointments, gels, suspension,emulsions, creams, lotions, powders and aqueous solutions. Glucan may beformulated with one or more carriers or excipients as are well known inthe pharmaceutical art (see, for example, Remingtons PharmaceuticalSciences, 17th Edition, Mack Publishing Company, Easton Pa., Ed Osol, etal, which is incorporated herein by way of reference).

[0089] Examples of carriers and excipient substances are organic orinorganic substances which are suitable for enteral (for example, oralor rectal), parenteral (for example, intravenous injection) or local(for example, topical, dermal, ophthalmic or nasal) administration andwhich do not react with the glucan, for example, water or aqueousisotonic saline solution. lower alcohols, vegetable oils, benzylalcohols, polyethylene glycols, glycerol triacetate and other fatty acidglycerides, gelatin, soya lecithin, carbohydrates such as lactose orstarch, magnesium stearate, talc, cellulose and vaseline.

[0090] Formulations may include one ore more preservatives, stabilizersand/or wetting agents, emulsifiers, salts for influencing osmoticpressure, buffer substances, colourants, flavourings and/or perfumes.

[0091] Glucan may be formulated into sustained release matrices whichliberate glucan over time providing what may be regarded as a depoteffect. Glucan in the form of a gel, as produced according to anembodiment of the aforementioned process, may be directly used as atopical pharmaceutical product or formulated with appropriate carriersand/or excipients.

[0092] In a further embodiment this invention is directed to a glucancomposition which consists essentially of branched β-(1,3)(1,6)-glucan,and which is free or essentially free of unbranched β-(1,3) glucan andnon-glucan components. Reference to “essentially free” is to beunderstood to refer to less than about 2% unbranched β-(1,3) glucan,more specifically less than about 0.5% unbranched β(1,3) glucan.

[0093] These glucan formulations may comprise glucan in microparticulateform, soluble form or as a gel, optionally formulated or in associationwith one or more pharmaceutically acceptable carrier or excipients asherein described.

[0094] Glucan containing formulations or compositions for therapeuticpurposes may contain from about 0.01% to about 30% (w/w). such as fromabout 0.1% to about 5%, more particularly from about 0.2% to about 1%,even more particularly from about 0.25% to about 0.5% (w/w). Theseamounts may be regarded as therapeutically effective amounts.

[0095] It has surprisingly been found by the inventors that Sc-glucan,whether produced according to this invention or by prior art processesmay be used in a range of hitherto unsuspected and undescribedtherapeutic applications. These applications include the treatment ofulceration or bone fracture, or the prevention/treatment of ultravioletlight induced skin damage.

[0096] In a further aspect this invention is directed to the use ofglucan for the manufacture of a medicament for the treatment of skinulceration or bone fracture, or the implantation/fixation of orthopaedicdevices, or prevention/treatment of ultraviolet light induced skindamage.

[0097] In a further aspect this invention is concerned with the methodfor the treatment of skin ulceration or bone fracture, or theimplanation/fixation of orthopaedic devices, or prevention/treatment ofultraviolet light induced skin damage, which comprises administering toa subject glucan in association with one or more pharmaceutically orveterinarily acceptable carriers or excipients.

[0098] In a still further aspect of this invention, there is provided anagent for the treatment of dermal skin ulceration, the enhancement ofrepair of bone and connective tissue, or the implanation/fixation oforthopaedic devices, or the prevention/treatment of ultraviolet lightinduced skin damage, which agent comprises glucan in association withone or more pharmaceutically or veterinarily acceptable carriers orexcipients.

[0099] In these novel therapeutic uses of glucan, an effective amount ofglucan is utilised. What constitutes an effective amount will depend onthe particular condition being treated, mode of and form ofadministration, and like factors. Generally, a composition or medicamentwill contain glucan in an amount from about 0.05% (w/w) to about 30%(w/w), such as 0.1 to 5% (w/w), more particularly from about 0.3% toabout 1% (w/w), even more particularly from about 0.25% to about 0.5%(w/w).

[0100] A particularly advantageous therapeutic application for glucan(such as microparticulate, soluble or gel forms manufactured by any ofthe aforementioned methods, or produced by prior art methods) accordingto the present invention is in the treatment of dermal ulceration. It isknown that β-1,3-glucan will promote healing in full-thickness,surgically-created skin wounds in animals and humans with nodysfunctional healing, That is, the topically- or parenterally-appliedglucan is able to accelerate the healing response in superficial woundswith normal healing mechanisms. It generally is assumed that glucanachieves this through activation of wound macrophages. Macrophages arecritical cells in the healing process, producing a range of cytokinesand growth factors which initiate the various components of the healingcascade—viz. fibroplasia, collagen production, angiogenesis,epitheiialisation and collagen cross-linking. The macrophage plays a keymodulatory role in this process, both initiating the process and helpingto ensure that the process proceeds in a co-ordinated and integratedmanner. It is assumed that a primary effect of the glucan is to producea temporal acceleration of the healing cascade.

[0101] Dermal ulcers typically are chronic wounds which have a quitedifferent set of physiological properties operating within the wound,compared to acute surgical wounds. Whereas the physiology of the healingprocess is well described for acute surgical wounds, it is ill definedfor chronic ulcers. Ulcers typically show poor to negligible healingbecause of either constant irritation or pressure (such as decubitusulcers or pressure sores) or restricted blood supply (such as inindividuals with arterial ischaemia or venous thrombosis) or infection(such as ‘tropical’ ulcers) or nerve damage (‘neurotrophic’ ulcers) ordiabetes. Ulcers have varying pathologies, and the underlying causes,where known, may be quite distinct. Various types of ulcers which may betreated according to this invention include those associated withphysical trauma (radiation, thermal burns, decubitus, insect bites),impaired blood flow (arterial, venous), infection (bone, pyogenic,synergistic gangrene, syphilis, tuberculosis, tropical diseases, fungaldiseases), neoplasia (primary skin tumour, metastases, leukemia) andneurotrophic lesions (spinal cord lesions, peripheral neuropathies).

[0102] Ulcers associated with dysfunctional healing vary greatly inseverity, from superficial wounds extending into the dermis and having asurface area of approximately 1-2 cm² up to wounds extending throughdermis, subcutaneous tissue and muscle and forming depressions andcavities with volumes of approximately 500 cm³. The larger ulcers inparticular can be debilitating and restrictive and require intensive andexpensive therapy to manage. Control of wound sepsis, regular wounddebridement, regular dressings, hypostatic drainage and correctivesurgery are just some of the standard current therapies. However,currently available ‘best-practice’ wound management therapies are notuniformly successful, take considerable lengths of time to producebeneficial results, are associated with poor rates of patientcompliance, generally are expensive, and are associated with a highincidence of ulcer recurrence. It has been noted by Margolis (J.Dermatological Surgery (1995) 21(2) 145-148) that: “a paucity of dataexists that adequately addresses the efficacy of any topical agent forthe treatment of pressure ulcers”.

[0103] It can be seen therefore that in view of the high incidence ofulcers in the community and the cost to the community of treatment,there is an urgent need to develop improved therapies. Ideally, such atherapy should have a high rate of success, be convenient to use andproduce a clinic response quickly in order to facilitate patientcompliance and preferably be inexpensive.

[0104] A particular difficulty in devising a uniformly successfultherapy which may be an improvement on current treatment modalities isthe non-unifomity of the different types of ulcers where both theunderlying aetiologic disease processes and the pathophvsiology withinthe wounds show considerable variation. Confounding this variability, isthe general poor understanding of which of the different components ofthe healing response is dysfunctional and therefore contributing theprimary pathology of the dysfunctional healing response. So thatsuccessful antagonism of dysfunction of any particular part of thehealing cascade in one ulcer type may not necessarily be successful inanother ulcer type. In particular there is no evidence that local woundimmune suppression or macrophage dysfunction are key pathologicalfeatures or that enhancement of local immune mechanisms within suchulcers would result in enhanced healing responses as is seen inuncomplicated surgical skin wounds with no dysfunctional healingresponses.

[0105] Thus it was entirely unexpected to find that topical applicationof glucan to decubitus, venous stasis and arterial ischaemic ulcersinduced rapid and potent healing responses in those wounds. This wasunexpected (a) because the primary causative factor of these ulcer typesis impaired blood supply and there is no evidence to suggest that thiswould be responsive to antagonism by an immune stimulant, and (b)because even where it might be possible to promote the healing response,the impaired vasculature to the wound could be expected to impede thehealing response as is observed with current treatment modalities. Thebeneficial effect of glucan in these ulcer types is even more remarkablegiven that a complete healing response can be achieved in the absence ofother supportive therapies such as sepsis control, hypostatic drainageand correction of the primary cause.

[0106] The treatment of decubitus ulcers and venostasis ulcers areparticularly preferred according to this invention, although theinvention is not limited to the treatment of these ulcer conditions.

[0107] Decubitus ulcers arise through multiple mechanisms. They are adisastrous complication of immobilization. They may result from shearingforces on the skin, blunt injury to the skin, drugs and prolongedpressure which robs tissue of its blood supply. Irritative orcontaminated injections and prolonged contact with moisture, urine andfaeces also play a prominent role. Diminished blood circulation of theskin is also a substantial risk factor. The ulcers vary in depth andoften extend from skin to a bony pressure point. Treatment is difficultand usually prolonged. Surgical techniques are at present the mostimportant means of achieving optimal healing.

[0108] Approximately half of venous ulcers are associated withincompetent perforating veins in the region of the ankle, and constitutea long term problem in many immobile patients. Ulceration is rarely amanifestation of primary varicose veins but is virtually alwaysassociated with incompetence of the popliteal venous valve. Venostasisulcers are most often just proximal or distal to the medial malleolus(bony ankle joint) and often develop at sites of minor trauma or skininfections. Scarring and secondary infection all impair healing and makerecurrences common if healing does occur. The natural history of venousulceration is cyclic healing and recurrence.

[0109] In the case of decubitus ulcers, the glucan preferentially isapplied in the form of a powder (microparticulate glucan) or in a creamor ointment base (microparticulate, soluble or gel forms of glucan).Application is generally daily and may continue for a time periodsufficient for ulcer healing, such as seven to twenty eight days. It isobserved that the response to the glucan therapy is apparent clinicallywithin 2-3 days with evidence of fresh granulation and epithelialgrowth. The length of time required to heal ulcers will vary accordingto a number of factors such as ulcer size, degree of wound sepsis andhost nutritional state. Typically wound volume is reduced by 50% within2-3 weeks with complete or near-complete wound closure effected by 4-6weeks after commencement of glucan therapy. It is noteworthy that mostof the decubitus ulcers in which glucan effects a healing response havebeen refractory to standard therapy including a wide range of topicalpreparations and wound dressings for periods up to 7 years.

[0110] In a similar manner, application of microparticulate, soluble orgel forms of glucan to venostasis and arterial ischaemic ulcers promotesulcer healing. As with the decubitus ulcers, treatment of these ulcerswith glucan leads to a clinical response in the wound within 2-3 daysfollowing the start of glucan therapy with such evidence of healing asthe appearance of fresh granulation tissue and less detritus leading toa cleaner appearance in the wound. Glucan in the form of a powder,cream, lotion, ointment or gel may be topically applied to the ulcersite daily until healing occurs. The chronic nature of the underlyingvascular disorder in these cases means that the predisposition to formsuch ulcers remains with the patient. It may be necessary therefore tocontinue glucan therapy on a long term basis to prevent recurrence.

[0111] It can be seen therefore that it is an entirely unexpectedobservation that glucan is able to promote the healing processed in skinulcers where the individual components of the healing process areessentially normal but are unable to antagonize the dysfunctional causesuch as inadequate blood supply, inadequate venous drainage, excessivetissue oedema, infection, constant pressure or other diverse causes.

[0112] It is observed that application of glucan to ulcers as describedabove produces a high rate of therapeutic response. Skin ulcers whicheither are unresponsive or poorly responsive to conventional woundmanagement practice, typically respond within several days to treatmentwith glucan leading in a high proportion of cases to complete healingwithin several weeks of treatment. It is found that the glucan iseffective in the treatment of ulcers when applied locally to the woundin various forms such as a powder, gel, cream, or dressing such as agauze bandage or colloidal material, or any other composition generallyknown to those skilled in the art of pharmaceutical formulation.

[0113] In a related aspect the treatment of ulcers which respond toconventional therapies (such as normal dressings and ointments) may beaccelerated with glucan administration.

[0114] Another unsuspected therapeutic application for glucan (such as,microparticulate, soluble or gel forms manufactured by any of theaforementioned methods, or other processes known in the art) accordingto the present invention is in the treatment of bone fracture. Therepair of fractured bone characteristically is accomplished by a repairprocess which basically is in common with that observed in soft tissuessuch as skin but which differs in some important aspects. In bone, animportant early step in the repair process is the formation of a fibrousstructure known as a callus which bridges the fractured site providing aframework for the repair process and assuring a decree of immobilizationof the fracture site. In due course the callus becomes mineralized,providing continuity with the uninjured bone and undergoes a degree ofremodelling to approximate the original shape of the bone. According tothis aspect of the invention the application of glucan to the site ofinjury enhances the rate of repair of injured bone thus facilitatingfracture treatment. It is observed that the effect of such treatment isearlier induction of the callus formation and earlier organization ofthe connective tissue within that callus to provide a strong fibrousmatrix. The result of this is the establishment of an immobilizingcallus at an earlier time with the important clinical effect of reducingthe risk of dissociation of the fractured edges of the bone. This is ahighly desirable effect in both animals and humans because anydisruption to the fracture site can predispose to delayed healing.Disruption at the fracture site remains a problem, even where methods ofphysical immobilization through such mechanical means as rigid splints(such as casts, bandages, etc.), or implants (such as pins, screws, etc)are used. While it is found that the process of mineralization is notappreciably enhanced by the glucan treatment, it is found that theeffect of glucan in accelerating the callus phase has the effect ofreducing the overall time to complete mineralization.

[0115] The glucan preferably is applied directly to the site of boneinjury in a form which will maximize the retention of the glucan at thesite of the fracture. Slow release formulations are well known in theart and are preferably used in these applications. It is found that theviscous gel formed by the embodiment disclosed in this invention wherebya highly alkaline soluble glucan solution at a concentration of greaterthan 15 mg/mL (from about 15 mg/ml to about 500 mg/ml, more preferablyfrom about 15 mg/ml to about 30 mg/ml) is adjusted to pH 7.5 (Example 4)is a preferred form. This form is sufficiently viscous and non-misciblewith blood and tissue fluids to remain at the site of application forperiods up to 48 hours. An additional advantage of this gel form is thatit is sufficiently tractable to be able to be injected through finegauge needles. In this form, the glucan can be administered by injectionto fracture sites where the fracture is reduced without the need forsurgical exposure of the bone. Alternatively, the gel can beadministered to the fracture site during open surgical reduction offractures.

[0116] The potential usefulness of glucan treatment for human bonefractures has been evidenced in an animal model by the inventors. Therat is a standard model used in experimental medicine for bone fractureresearch and generally is regarded as directly predictive of humantherapy (Bak et. al. 1992). In this animal model the inventors haveestablished that injection of 2 mL of 15 mg/mL soluble glucan in a gelform at the site of a fractured femur resulted in accelerated healingwhen compared with non-treated fractures as evidenced by increasedtensile strength of the partially healed bones at 12 days (Example 10).

[0117] It can thus be readily envisaged that glucan, being non-toxic andphysiologically acceptable, may find wide application in fracturetreatment in human and animal medicine. For example, a single bolusinjection or application of glucan at the site of fracture will promotehealing and increase tensile strength of the healed bone.

[0118] A further unexpected therapeutic benefit is that glucan enhancesthe fixture of devices such as pins, screws, artificial joints andprostheses fixed or implanted into or onto bone. It is observed that theapplication of glucan (such as by local application of a powder or gel,or by injection) at the site of fixation of the device enhancessignificantly the local inflammatory process which occurs in response tothe contact of the device with bone and generally is an integral part ofthe strength of the bond between the bone and the device.

[0119] A particular therapeutic indication for glucan (eithermicroparticulate or soluble forms manufactured by any of theaforementioned methods or by prior art methods) according to the presentinvention is in the treatment of injured connective tissues such astendons and ligaments which has not previously been described orsuggested. Such tissues are typically densely fibrous because they aresubjected to relatively high stress loads. These injuries include by wayof example but are not limited to (a) acute or chronic inflammationassociated with over use or strain or trauma, such conditions typicallybeing associated with sporting injuries or the syndrome known asRepetitive Strain Injury or excessive or abnormal stress, and (b)surgery, in particular where the tissue is dissected or transected. Itis known that injuries of this kind in such tissues typically are slowto heal, due in part to the relative difficulty of totally resting theinjured tissue because of their load bearing functions, but due largelyto the characteristically lower level of activity of all aspects of thetissue healing cascade compared to that which is seen in soft tissues.An important cause of this comparatively lower level of tissue repairactivity in tendons and ligaments is a more limited blood supplycompared to most soft tissues. It is found that application of glucan tothe injured tendon or ligament either at the time of acute injury suchas following surgery or external trauma, or with chronic injury such aschronic inflammation will promote both the rate of onset and the levelof the healing response in these tissues, leading in the case of surgeryto earlier return of normal strength and function and in the case ofinflammation to earlier resolution of the inflammatory process. Theglucan may be directly injected into the injured tendon or ligament.Although it has been described that glucan is a potent enhancer of woundrepair in dermal tissue in healthy tissues,it is not apparent from thatknowledge that glucan has the ability to effect enhanced resolution ofchronic inflammatory processes or of enhancing repair processes intissues with limited blood supply or where the normal rate of repair isknown to be relatively slow.

[0120] A further unsuspected therapeutic indication of glucan is theprevention/treatment of ultraviolet light-induced skin damage whichresults from exposure to the sun.

[0121] It is well described that ultraviolet light exposure causesdamage to skin, particularly long term exposure to sunlight. This isparticularly the case with Caucasians who have light skin colourationwhich predisposes them to photo-ageing and development of certain typesof skin cancers. Both of these problems are prominent within mostWestern communities.

[0122] The detrimental effects of sunlight are due primarily to itsultraviolet light spectrum (UV-A and UV-B). UV-B acts principally withinthe epidermis and rarely penetrates deeper than the uppermost layers ofthe dermis, while the longer wave-length UV-A penetrates through thedermal layers. The major detrimental effect of ultraviolet light isdamage to proteins, particularly DNA and RNA where it results in dimerformation. Most of these dimers are repaired within several hoursalthough a small number are either not repaired or are mis-repaired andthe accumulation of these mis-repairs over a lifetime is thought to be amajor contributing factor to the development of skin carcinogenesis inchronically sun-exposed individuals.

[0123] The two principal outcomes of this damage to proteins in the skinis acute cell damage and mutagenicity. Cell damage is evidencedclinically in the acute phase by the symptoms referred to generally as‘sun-burn’ which include erythema (reddening) and oedema and in thelong-term phase by symptoms referred to generally as ‘photo-ageing’which include skin thickening and wrinkling; mutagenicity is evidencedby skin cancer development. A further effect of ultraviolet light whichis not clinically apparent is immune depression. Skin has a rich networkof immune cells that are equally sensitive to the detrimental effects ofultraviolet light as are other skin cells and exposure to ultravioletlight leads to temporary dysfunction of these cells. This dysfunction isrepaired generally within 2-3 days but in this period the skin showsreduced immune capacity such as antigen-presentation. With repeatedultraviolet light exposure such as might be expected in individuals witha lifetime exposure to sunlight, the sun-exposed skin has chronicallyreduced immune function. It is likely that this predisposes to thedevelopment of skin cancer through reduced immune surveillance capacitywithin skin. However, the relative contributions that each of thedifferent effects of ultraviolet light (viz. immune depression, chronicdermal and epidermal cell injury, mutagenicity) has in skin cancerdevelopment and photo-ageing remains unknown.

[0124] It has been found surprisingly by the inventors that glucanapplied topically to skin either following or concominant withultraviolet light leads to substantial protection of the skin fromultraviolet light-induced skin damage.

[0125] This has been found in experiments conducted with a standard,hairless mouse strain used as a model to study solar damage to humanskin (see, for example. Canfield et al 1985). In this model the mice areexposed daily for 10 weeks to a minimal erythema dose of mixedultraviolet light which simulates the toxic effects of sunlight on skin.Each daily exposure of ultraviolet light induces a mild erythema andoedema lasting up to about 24 hours and which mimics in appearance amild ‘sun-burn’ in humans. With continued irradiation treatment thison-going damage is reflected in progressive thickening of the skin whichhistologically mimics the hyperkeratinisation and elastosis associatedwith photo-ageing in chronically sun-expose skin in humans.Pre-malignant tumours begin to appear within several weeks of completionof the ultraviolet light treatment regime. Over the ensuing 6-12 monthsthere is progressive development of pre-malignant and malignant tumours,the histology and behaviour of which closely mimic the actinic keratosesand pre-malignant and non-melanona skin cancers that develop in humansin response to sunlight.

[0126] The inventors have found that soluble glucan applied to the skindaily immediately following ultraviolet irradiation provides significantprotection from both the acute toxic effects (evidenced by discerniblylesser skin erythema on each morning following the previous day'sirradiation) and the chronic photo-ageing effects (evidenced bysignificantly thinner skin). This effect is particularly unexpectedgiven that β-1,3-glucan is not previously known to protect tissues fromdirect cytotoxic damage and that there is no existing data that eitherconfirms or suggests that β-1,3-glucan antagonises the cytochemical andhistopathological lesions that are consequent to acute or chronicultraviolet irradiation. The ability of glucan in this model toantagonise the acute toxic and chronic photo-ageing effects ofultraviolet irradiation offers a novel and important means of protectionof human skin from the damaging effects of sunlight.

[0127] It also has been found by the inventors that soluble glucanapplied topically to human skin immediately following exposure tosunlight affords protection from the acute erythemal effects of theultraviolet light.

[0128] It further is found in the hairless mouse model that the glucanaffords considerable protection from the development of skin cancers(see FIG. 1 hereafter). The majority of tumours at this early stage arebenign sessile-based papillomas, as expected: transformation of aproportion of these to more malignant intermediate forms culminating insquamous cell carcinomas is anticipated at a later stage.

[0129] Accordingly, glucan may find wide applications in amelioratingthe effects of sunlight in the human population. In this regard, thebeneficial effect of glucan is obtained if it is applied either priorto, during or following sunlight exposure. To this end, it may beformulated into sunscreen formulations or into after-sun or in generalcosmetic formulations such as lotions, creams and gels. The particularbenefits to be gained from the use of Sc-glucan include the following:(a) amelioration of the acute toxic effects of sunlight on skin (‘acutesunburn’); (b) amelioration of the chronic effects of sunlight on skinwhich collectively are known an photo-ageing and include symptoms suchas hyperkeratinisation, skin thickening, elastosis and wrinkling; (c)amelioration of the development of sunlight-induced skin carcinogenesis.

[0130] It is to be understood that the novel therapeutic uses for glucanherein described are not limited to glucan produced by the processesdescribed herein, although this material is preferred. Any prior glucanmaterial such as those described by Hassid et al, Di Luzio et al,Manners et al and Jamas et al (U.S. Pat. Nos. 5,028,703, 5,250,436,5,082,936 and 4,992,540) may be used. Preferably the glucan is Sc-glucan

[0131] This invention will now be described with reference to thefollowing non-limiting examples which illustrate various embodiments ofthe invention.

[0132]FIG. 1 depicts dorsal skin fold thickness measurements in micesubject to U.V. irradiation over 6 weeks, which mice are treated withglucan (-□-) or treated with a non-glucan base lotion (-

-).

EXAMPLE 1

[0133] Microparticulate glucan is prepared as follows:

[0134] A 400 g sample of commercial Saccharomyces cerevisiae in dry formis added to four litres of 4% w/v sodium hydroxide and heated to 100° C.for one hour with vigorous stirring. The suspension is allowed to coolto between 45° C. and 50° C. before the lysed yeast cells are separatedfrom the alkaline hydrolysate by centrifugation at 800 g for tenminutes. The lysed yeast cells are resuspended in a fresh batch of threelitres of 3% w/v sodium hydroxide and boiled for 15 minutes. Followingseparation by centrifugation, the lysed yeast cells are resuspended in afresh batch of two litres of 3% w/v sodium hydroxide and boiled for 15minutes followed by standing at 70° C. for 16 hours. Followingseparation by centrifugation, the lysed yeast cells are resuspended inwater and boiled for 10 minutes. The latter step is repeated once.Following centrifugation, the lysed yeast cells are resusended in afresh aliquot of 2 L water, the pH adjusted to 4.5 by the addition ofphosphoric acid and the suspension then boiled for thirty minutes. Fivehundred mL of chloroform then is added and the suspension subjected tovigorous agitation for ten minutes, following which the suspension isallowed to settle for 10 minutes in a separating funnel. The suspensionseparates into three distinct phases, a lower organic phase, an upperaqueous phase, and an interface between these two phases which is greycoloured. The lower chloroform phase plus a greyish intermediate phaseare discarded, leaving an aqueous phase which is collected and exposedas before to a fresh batch of 500 mL of chloroform. The final aqueousphase is collected and boiled for 10 minutes to remove any residualchloroform and then dried using a spray-drier.

[0135] Analysis of the aqueous phase showed that it contained onlybranched β-(1,3)(1,6) glucan in the ratio of 95 to 98% β-1,3:2 to 5%β-1,6 linkages. The organic phase is slightly opaque and contains lipidsbut no glucan. The intemediate phase (interface) contains unbranchedβ-(1,3) glucan (98 to 100% β-1,3:0 to 2% β-1,6) associated withinchitin, protein and other non-glucan contaminents. Biological testsshowed that the branched glucan was significantly more biologicallyactive than unbranched β(1,3) glucan in a wound healing test.

[0136] The chemical composition of glucan produced according to thisinvention is set forth in Table 7. TABLE 7 Chemical composition ofSc-glucan produced by the process of the present invention. % (byweight) Glucose¹ >98 Mannan¹ <0.2 Protein² <0.5 Glycogen³ <0.5 Chitin¹<0.3 Lipid⁴ not detectable Glycosidic linkages:⁴ β-1,3 96-97 β-1,6 3-4

[0137] It is clear from this analysis that the end-product is a branchedβ(1,3)(1,6) glucan that is substantially pure, containing only traceamounts of impurities, and containing about 2 to 3% β-1,6 linkages.

EXAMPLE 2

[0138] Microparticulate Sc-glucan is prepared as follows:

[0139] A 400 g sample of commercial Saccharonzyces cerevisiae in dryform is added to four litres of 4% w/w sodium hydroxide and heated to100° C. for one hour with vigorous stirring. The suspension is allowedto cool to between 45° C. and 50° C. before the lysed yeast cells areseparated from the alkaline hydrolysate by centrifugation at 800 g forten minutes. The lysed yeast cells are resuspended in a fresh batch ofthree litres of 3% w/v sodium hydroxide and boiled for 15 minutes.Following separation by centrifugation, the lysed yeast cells areresuspended in a fresh batch of two litres of 3% w/v sodium hydroxideand boiled for 15 minutes followed by standing at 70° C. for 16 hours.Following separation by centrifugation, the lysed yeast cells areresuspended in water and boiled for 10 minutes. The latter step isrepeated once. Following centrifugation, the lysed yeast cells areresusended in a fresh aliquot of 2 L water, the pH adjusted to 4.5 bythe addition of hydrochloric acid and the suspension then boiled for tenminutes. Five hundred mL of chloroform then is added and the suspensionsubjected to vigorous agitation for ten minutes, following which thesuspension is allowed to settle for 10 minutes in a separating funnel.The lower chloroform phase plus a greyish intermediate phase arediscarded, leaving an aqueous phase which is collected and exposed asbefore to a fresh batch of 500 mL of chloroform. The final aqueous phaseis collected and boiled for 10 minutes to remove any residual chloroformand then dried using a spray-drier.

[0140] The chemical composition of glucan produced according to thisinvention is set forth in Table 8. TABLE 8 Chemical composition ofSc-glucan produced by the process of the present invention. % (byweight) Glucose¹ >98 Mannan¹ <0.2 Protein² <0.5 Glycogen³ <0.5 Chitin¹<0.3 Lipid⁴ not detectable Glycosidic linkages:⁴ β-1,3 98-99 β-1,6 1-2

[0141] It can be seen that compared to the end-product material obtainedin Example 1, this material has has a similar degree of purity but hasslightly fewer β-1,6-glucan linkages indicating a lesser degree ofside-branching.

EXAMPLE 3

[0142] A protocol for the preparation of minimally-polymerised, solubleSc-glucan according to the present invention is as follows.

[0143] Microparticulate Sc-glucan is produced as detailed in Example 2.Ten g of this material is suspended in 100 mL sterile 5% NaOH solutionand stirred gently for two hours at 5° C. (giving a pH around pH 13).The suspension then is diluted 1:1 in sterile, distilled water and thenfiltered through a 1 u membrane to remove undissolved particulatematerial. The pH of the filtered solution then is adjusted to 10 by theaddition of 5M HCl and then dialysed against 2 L distilled water (pH 10)in a Pelicon system using a 10,000 D limiting membrane. The solutionthen can be sterilised by passage through a 0.45μ membrane and the pH ofthe solution may be adjusted as desired. The soluble glucan so producedis useful as a pharmaceutical product.

[0144] Gel permeation chromatography (Waters Styragel HR 5® column;effective molecular weight range of 10×10⁴ to 4.0×10⁶ daltons) of thesoluble glucan showed the material was essentially homogenous with avery narrow molecular weight spread, having an average molecular weightof 140,000 daltons and a polydispersity index of 2.564. In thisdetermination the solvent is DMSO and the column flow rate is 1ml/minute.

EXAMPLE 4

[0145] A protocol for the preparation of polymerised, soluble glucanaccording to the present invention is as follows.

[0146] Microparticulate Sc-glucan is produced as detailed in Example 2.Fifteen g of this material is suspended in 100 mL sterile 5% NaOHsolution and stirred gently for two hours at 5° C. The suspension thenis centrifuged at 1000 g to remove undissolved particulate material. ThepH of the solution then is adjusted to 10 by the addition of 5M HCl andthen dialysed against 2 L distilled water (pH 10) in a Pelicon systemusing a 10,000 D limiting membrane. The pH then is adjusted to 7.5 bythe further addition of hydrochloric acid producing a viscous gel whichis useful as a pharmaceutical product.

[0147] Gel permeation chromatography showed the material was essentiallyhomogenous with a very narrow molecular weight spread, having an averagemolecular weight of 320,000 daltons and a polydispersity index of 2.2.

EXAMPLE5

[0148] A model of delayed wound healing was developed in rats to testthe ability of microparticulate Sc-glucan to promote wound healing indysfunctional wounds. The breaking strength of seven day-old skin woundsin inbred young adult laboratory rats is determined as outlined earlierbut the rats in this case are treated with drugs intended to depress thehealing response. This is achieved by daily treatment from the time ofwounding with a combination of prednisone (1 mg/kg), cyclosporin A (5mg/kg) and azothioprine (2 mg/kg). This triple drug therapy provides arange of depressive effects on macrophages, lymphocytes and vascularendothelium.

[0149] Table 9 summarizes the results of the use of Sc-glucan in thismodel. The effect of the triple drug therapy was to reduce significantly(p<0.01) the breaking strength of the wound at seven days. A singleapplication of 1 mg of microparticulate Sc-glucan (per 5 cm linearlength skin wound) produced by the process of the present inventionsuccessfully antagonized the depressive effect of the triple drugtherapy, returning the breaking strength of the wound to that seen innormal immunocompetent rats. TABLE 9 Effect of topical Sc-glucan therapyon the breaking strength of skin wounds in rats with and withoutdrug-induced depressed wound healing. Wound breaking Group Drugtreatment Glucan treatment strength (g) (mean) 1 None None 422 2 YesNone 275 3 Yes Yes 442

EXAMPLE 6

[0150] Glucan Formulations

[0151] A topical preparations for human and veterinary applications wereprepared from the following components: TOPICAL CREAM β-1,3-glucan(microparticulate) BP 1 mg/g Zinc stearate BP 3 mg/g Cetomacrogol 1000BP 20 mg/g Cetostearyl alcohol BP 80 mg/g Phenoxyethanol BPC 1973 5 μL/gGlycerol BP 60 mg/g Arachis oil BP 40 mg/g Purified water BP to 1 g

[0152] This formulation may be referred to as Formulation #1.

[0153] A powder for topical application was prepared from the followingcomponents: TOPICAL POWDER β-1,3-glucan (microparticulate) 100 mg/gMaize corn flour BP 900 mg/g

[0154] This formulation may be referred to as Formulation #2.

[0155] A topical cream was prepared by mixing the following components:TOPICAL CREAM Paraffin oil  80 ml Olive oil  60 ml Anhydrous lanolincetomacrogol 1000  60 g Stearic acid Cetostearyl alcohol  58 g Glycerylmonostearate phenoxyethanol  60 g Oleic acid glycerol  25 ml Water 1200ml Triethanolamine  27 ml Soluble glucan of Example 3  20 ml

[0156] This formulation may be referred to as Formulation #3 andprovides a cream containing 5 mg soluble glucan per g.

[0157] Formulations #1 to #3 were varied by incorporating glucan in theform of a gel. These may be referred to as Formulations #1A to #3A.

EXAMPLE 7

[0158] A decubitus ulcer was treated successfully in a human patientusing Formulation #1.

[0159] The patient was a ninety year old male stroke victim who had beenhospitalized for ten years and who was essentially bed-ridden. Adecubitus ulcer had developed on the right buttock in 1986 and persisteddespite regular medical and nursing attention. By 1988 the ulcer hadgrown to a diameter of 8 cm and to a depth of 4 cm. Conventionaltreatments consisting of regular wound cleansing, application ofprotective dressings and body positioning to minimize pressure to theulcer had failed to halt the progressive deterioration of the ulcer.

[0160] Treatment with Sc-glucan was commenced and involved topicalapplication using Formulation #1. Daily topical treatments were carriedout for one week and then ceased. Two weeks after treatment the ulcerwas totally healed; epithelialization was complete and there was novisible scar formation.

EXAMPLE 8

[0161] A patient (Mr G W) suffering from persistent leg ulcers wastreated with glucan (Formulation #1).

[0162] The patient was a fifty three year old male who suffered asporting injury which included a fractured ankle. Following this injurythe ankle was reconstructed twice. After the second reconstruction thewound did not heal and four venostasis ulcers developed despite the useof systemic and topical bactericides and antibiotics.

[0163] Following five successive daily applications of the glucancontaining formulation 1 wound healing cream of Example 5 to three ofthe ulcers, one originally measuring 3.8 cm×1.9 cm completely healed inten days; one measuring 10.2 cm×3.8 cm was reduced to 6.3 cm×1.3 cmduring the ten day treatment period; and a further ulcer measuring 3.8cm×1.9 cm was reduced to 2.5 cm×1.2 cm. The treatment was recommenced onthe tenth day and after two further cycles of treatment comprising creamapplication for seven days and no treatment for seven days the lattertwo ulcers completely healed after four weeks. Treatment of the fourthulcer (10 cm×9 cm) involving two exposed tendons and extensive tissuenecrosis was commenced shortly thereafter. After ten days of dailytreatment, there was clear evidence of epithelial regrowth andgranulation tissue leading to coverage of the exposed tendons bygranulation tissue and overall reduction in wound size to 8 cm×7 cm.

[0164] The patient had never observed such positive results from anyprevious treatment.

EXAMPLE 9

[0165] The posterior aspect of the forearm of a six year oldthoroughbred stallion was severely traumatized in a fight with anotherstallion creating a deep cavity with an external hole some 40 cm×20 cmin area. Initial treatment was irrigation with disinfectant andantibiotic solutions but after several days the severity of the injurybecame more apparent and appeared to be worsening. There was extensiveand deep sloughing occurring with necrosis of deep tissues includingligament and tendons and associated muscle masses—some tendon remnantswere present as unhealthy looking strands and the animal could not bearweight. The affected area was treated at that time by topicalapplication of Formulation #1 of Example 5.

[0166] There was an immediate and profound response to glucan treatment.

[0167] The sequence of the clinical response to treatment was asfollows:

[0168] 24 hr post-treatment: Necrosis lessened with reduction insuppuration.

[0169] 36 hr post-treatment: Marked improvement in appearance of woundwith tissue showing vitality.

[0170] 72 hr post-treatment: Whole area filling in rapidly with ligamentand tendon remnants being included in new tissue.

[0171] 96 hr post-treatment: General appearance of good rapid healthyhealing with peripheral epithelialization evident.

[0172] The wound ultimately completely closed after 12 days of treatmentand with minimal scarring.

[0173] The animal at that time was weight-bearing on all legs.

EXAMPLE 10

[0174] Four adult rats (male, Wistar, inbred) had their left femursbroken under anaesthesia using externally-applied force. The fracturesite was located by external palpation and a 21-gauge needle thenintroduced through the skin over the fracture site and positionedbetween the fractured ends of the femur. The fracture then wasimmobilised in the standard way by insertion of an intra-medullary pinthrough the knee joint to emerge through the femoral head. In two rats,2 mL of colloidal glucan produced as per Example 4 were injected intothe fracture site via the previously-positioned needle. In the other tworats, 2 mL of saline was injected instead of glucan.

[0175] The needle then was withdrawn and the rats allowed to recoverfrom the anaesthetic. Twelve days later the rats were killed, theintra-medullary pins removed and the fractured femurs isolated forvisualisation of the fracture site and determination of the strength ofthe healing response. In the two control (saline) rats, the fracturesite was contained within a rudimentary callus and was able to bedisplaced readily by torsion of the upper and lower femoral shafts. Inthe two glucan-treated rats, the callus was further advanced, beingfirmer and considerably greater force was required to displace thefractured ends of femur. It was concluded that the effect of the glucanhad been to accelerate callus formation, leading to a firmed bond of thefracture site at 12 days post-fracture.

EXAMPLE 11

[0176] A 50 year-old Caucasian male exposed an area of skinapproximately 4 cm×12 cm on the inner aspect of both forearms to directsunlight for a period of 40 minutes. Both areas were exposed underidentical conditions and both forearms had similar levels of skinpigmentation. Each exposed area was divided into 4 equal patches (4 cm×3cm) which were delineated by indelible ink. On each forearm, 1 gm ofsun-cream (SPF 10) was applied to one of the end patches prior tosun-exposure, the remaining patches were untreated at this time. Twohours following sun-exposure Sc-glucan (Formulation #3 from Example 6)was applied to the second patches, the third patch was left untreated,and 2 gm of Formulation #3 (Example 6) base without Sc-glucan applied tothe fourth patch. The order of treatment was reversed on each forearm.

[0177] The skin patches were examined 24 hours following sun-exposureand the degree of redness assessed visually by scoring 0, +, ++, +++ and++++. The results were as follows: untreated ++++ SPF 10 + cream baseonly ++++ glucan + cream base ++

[0178] The glucan effected considerable reduction of skin redness.Hence, glucan ameliorated the clinical response to sun damage.

EXAMPLE 12

[0179] Albino Skh:HR-1 hairless mice were irradiated daily with U.V.light for a period of 12 weeks. After each daily irradiation, mice werepainted with glucan cream of (Formulation #3), cream base alone oruntreated. Results are shown in Table 10. TABLE 10 Mean no. ofpre-malignant (papillomas, hyperkeratoses, kerato-acanthomas) andmalignant (carcinomas) in albino Skh: HR-1 hairless mice, following 12weeks ultraviolet irradiation painted with 0.1 ml of either cream baselotion or Sc-glucan (7 mg/day) and cream base each day. Mean no. skintumors per mouse Weeks Treatment no. mice 11 14 17 19 21 Cream base 20 01.7 ± 4.7 ± 7.6 ± 13.3 ± 8.3 only 2.7 3.9 3.9 Sc-glucan + 20 0.05 ± 0.05± 0.95 ± 1.7 ± 4.6 ± cream base 0.2 0.2 1.9 2.6 4.7*

EXAMPLE 13

[0180] Mice are exposed daily for ten weeks to a minimal erythemal doseof U.V. light which stimulates the toxic effects of sunlight on skin.Each daily exposure of U.V. light induces a mild erythema and oedemalasting up to 25 hours which mimmics a mild ‘sun-burn’ in humans. Micewere either treated with Formulation #3 after U.V. light exposure(group 1) or treated with base lotion containing no glucan (group 2). Atsix weeks notable skin thickening (and consequential wrinkling) wasobserved for group 2 mice. Mice of group 1 were largely protected fromthese effects. Erythema was not observed in group 1mice over thetreatment period. FIG. 1 depicts the results obtained in one test. After6 weeks, glucan treated mice (-□-) showed appreciably less skin foldthickness than untreated mice (-

-).

References

[0181] Bacon J S D, Farmer, V C, Jones D, Taylor I F, “The glucancomponent of the cell wall of baker's yeast (Saccharomyces cerevisiae)considered in relation to its ultrastructure”, Biochem. J., 114, 557-567(1969)

[0182] Bak B, Jensen K S, “Standardization of Tibial Fractures in theRat” Bone, 13,289-295 (1992)

[0183] Canfield P J, Greenoak G E, Reeve V E, Gallagher C H,“Characterisation of UV induced keratoancanthoma-like lesions inHRA/Skh-1 mice and their comparison with keratoacanthomas in man”,Pathology, 17(4), 613-616 (1985)

[0184] Cook J A, Holbrook T W, Parker B W, “Visceral leishmaniasis inmice: protective effect of glucan”. Journal of the ReticuloendothelialSociety, 27, 567-573 (1980)

[0185] Czop J K, Austen K F, “Generation of leukotrienes by humanmonocytes upon stimulation of their β-glucan receptor duringphagocytosis”, Proceedings of the National Academy of Sciences (USA),82, 2751-2755 (1985)

[0186] Di Luzio N R, Williams D L, McNamee R B, Edwards B F, Kitahama A.“Comparative tumor-inhibitory and antibacterial activity of soluble andparticulate glucan”. Int J Cancer. 24, 773-779 (1979)

[0187] Deimann, Fahimi. Journal of Experimental Medicine, 149, 883-897(1979)

[0188] Hassid W Z, Joslyn M A, McCready R M, “The molecular constitutionof an insoluble polysaccharide from yeast, Saccharomyces cerevisiae”,Journal of the American Chemical Society, 63, 295-298 (1941)

[0189] Kelly G E, Lui, W, “Accelerated wound healing in normal andimmunosuppressed animals”, Norvet Research Pty Ltd, 1994, Report G94003.

[0190] Maeda Y Y, Chihara G, “The effects of neonatal thymectomy on theantitumour activity of lentinan, carboxymethylpachymaran and zymosan,and their effects on various immune responses”, International Journal ofCancer, 11, 153-161 (1973)

[0191] Manners D J, Masson A J, Patterson J C, “The structure of aβ-(1,3)-D-glucan from yeast cell walls”, Biochem J, 135, 19-30 (1973)

[0192] Mansell P W A, Ichinose H, Reed R J, Krementz E T, McNamee R, DiLuzio N R, “Macrophage-mediated destruction of human malignant cells invivo”. Journal of the National Cancer Institute, 54, 571-580 (1975)

[0193] Niskanen, Cancer Research, 38, 1406-1409 (1978)

[0194] Patchen, Lotzova, Experimental Haematology 8, 409422 (1980)

[0195] Riggi S, Di Luzio N R, “Identification of a RE stimulating agentin zymosan”. American Journal of Physiology 200, 297-300 (1961)

[0196] Sherwood E R, Williams D L, Di Luzio N R, “Glucan stimulatesproduction of antitumor cytolytic/cytostatic factor(s) by macrophages”,Journal of Biological Response Modifiers, 5, 504-526 (1986)

[0197] Sherwood E R, Williams D L, McNamee R B, Jones E L, Browder I W.Di Luzio N R, “Enhancement of interleukin-1 and interleukin-2 productionby soluble glucan”, Internatioinal Journal of Immunopharmacology, 9,261-267 (1987)

[0198] Williams D L, Pretus H A, McNamee R B, Jones E L, Ensley H E,Browder I W, Di Luzio N R, “Development, physicochemicalcharacterization and preclinical efficacy evaluation of a water solubleglucan sulfate derived from Saccharomyces cerevisiae” Immunopharmacol,22, 139-156 (1991)

[0199] Williams D L, Cook J A, Hoffmann E O, Di Luzio N R, “Protectiveeffect of glucan in experimentally induced candidiasis”, Journal of theReticuloendothelial Society 23, 479-490 (1978)

[0200] Williams D L, Browder I W, Di Luzio N R, “Immunotherapeuticmodification of E. coli-induced experimental peritonitis and bacteremiaby glucan”, Surgery, 93, 448-454 (1983)

[0201] Williams D L, Sherwood E R, McNamee R B, Jones E L, Di Luzio N R,“Therapeutic efficacy of glucan in a murine model of hepatic metastaticdisease”, Hepatology, 5, 198-206 (1985)

[0202] Williams D L, McNamee R B, Jones E L, Pretus H A, Ensley H E,Browder I W, Di Luzio N R, “A method for the solubilization of a(1-3)-β-D-glucan isolated from Saccharomyces cerevisiae”, CarbohydrateResearch, 219, 203-213 (1991)

[0203] The references referred to herein are incorporated by reference.

1. A process for production of β-(1,3)(1,6)-glucan from a glucancontaining cellular source which comprises the steps of: (a) extractingglucan containing cells with alkali and heat, in order to remove alkalisoluble components; (b) acid extracting the cells of step (a) with anacid and heat to form a suspension; (c) extracting the suspensionobtained of step (b) or recovered hydrolyzed cells with an organicsolvent which is non-miscible with water and which has a density greaterthan that of water separating the resultant aqueous phase, solventcontaining phase and interface so that substantially only the aqueousphase comprising β-(1,3)(1,6)-glucan particulate material remains;wherein the extraction with said organic solvent provides separation ofglucan subgroups comprising branched β-(1,3)(1,6) glucan, andessentially unbranched β-(1,3) glucan which is associated with residualnon-glucan contaminents; and (d) drying the glucan material from step(c) to give microparticulate glucan.
 2. A process according to claim 1,which is a process for producing soluble glucan, wherein step (d) isomitted and the pH of the glucan material is raised so as to effectsolubilization of the glucan, and wherein the temperature of reaction isless than about 60° C.
 3. A process according to claim 1, which is aprocess for producing soluble glucan, wherein the particulate glucan ofstep (d) is suspended in an aqueous alkali solution so as to effectsolubilization of the glucan, and wherein the temperature of reaction isless than about 60° C.
 4. A process according to claim 2 or 3, whereinthe temperature of reaction is between about 2° C. and about 8° C. andwherein the soluble glucan has a polydispersity index suitable for useas a pharmaceutical product.
 5. A process according to claim 1 whereinthe acid used at step (b) is selected from acetic acid, formic acid,phosphoric acid and hydrochloric acid.
 6. A process according to claim 1wherein the pH of the acid of step (b) is from about 2 to about
 6. 7. Aprocess according to claim 1 wherein the organic solvent of step (c) isselected from chloroform, methylchloroform, dichloromethane,tetrachloroethane and carbon tetrachloride.
 8. A process according toclaim 7 wherein said solvent is chloroform.
 9. A process according toclaim 2 or 3 wherein the solution pH after glucan solubilisation isadjusted to the range of about pH 9 to about pH 10, and the resultantsoluble glucan is admixed with one or more pharmaceutically acceptablecarriers or excipients.
 10. A process according to claim 2 or 3 whereinthe solution pH after glucan solubilisaion is adjusted from about 7 toabout 8 so as to form a gel which optionally is admixed with one or morepharmaceutically acceptable carriers or excipients.
 11. Particulateglucan when produced according to the process of claim 1, optionally inassociation with a pharmaceutically acceptable carrier or excipient. 12.Soluble glucan when produced according the process of any one of claims2 to 4, optionally in association with a pharmaceutically acceptablecarrier or excipient.
 13. A glucan gel when produced according to theprocess of claim 10, optionally in association with a pharmaceuticallyacceptable carrier or excipient.
 14. A composition which consistsessentially of branched β(1,3)(1,6)-glucan and which is essentially freeor unbranched β(1,3) glucan, optionally in association with one or morepharmaceutically acceptable carriers or excipients.
 15. A glucancomposition according to claim 1, wherein said glucan ismicroparticulate, soluble in aqueous solution, or is the form of a gel.16. Use of glucan for the manufacture of a medicament for the treatmentof skin ulceration or bone fracture or the enhancement of fixation ofimplanted orthopaedic devices, or for the prevention/treatment ofultraviolet light induced skin damage.
 17. Use of glucan for thetreatment of skin ulceration or bone fracture or the enhancement offixation of implanted orthopaedic devices, or for theprevention/treatment of ultraviolet light induced skin damage.
 18. Amethod for the treatment of skin ulceration or bone fracture or theenhancement of fixation of implanted orthopaedic devices, or for theprevention/treatment of ultraviolet light induced skin damage whichcomprises administering glucan to a subject, optionally in associationwith one or more pharmaceutically, veterinarily or agriculturallyacceptable carrier or excipient.
 19. An agent for the treatment of skinulceration or bone fracture or the enhancement of fixation of implantedorthopaedic devices, or for the prevention/treatment of ultravioletlight induced skin damage which comprises glucan optionally inassociation with one or more pharmaceutically acceptable carriers orexcipients.
 20. Use of glucan according to claim 16 or 17 wherein saidglucan is produced according to any one of claims 1 to
 10. 21. A methodaccording to claims 18 wherein said glucan is produced according to anyone of claims 1 to 10.